Recombinant human SM-11044-binding receptor proteins exhibiting ligand-binding activities, and their use

ABSTRACT

Transformed cells designed to express a recombinant human SMBP at an elevated level to the extent that its ligand-binding activity can be measured, and cellular membrane fractions thereof; recombinant human SMBPs isolated from the transformed cells or the cellular membrane fractions thereof; a screening system for human SMBP agonists/antagonists characterized by utilizing the transformed cells, the cellular membrane fractions thereof or the isolated recombinant human SMBPs; and human SMBP agonists or antagonists obtainable by the screening system, are provided by deleting the polythymidine sequence from the base sequence of the 3′-untranslated region.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP99/04808 which has an Internationalfiling date of Sep. 6, 1999, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to recombinant human SM-11044((L)-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl)propyl]serinepyrrolidine amide hydrobromide)-binding receptor proteins exhibitingligand-binding activities (abbreviated as SMBP hereinafter), and theiruses. More particularly, it relates to transformed cells that aredesigned to express a recombinant human SMBP at an elevated level to theextent that its ligana-binding activity can be measured by deleting thepolythymidine sequence from the base sequence of the 3′-untranslatedregion, and cellular membrane fractions thereof, to recombinant humanSMBPs isolated from the transformed cells or the cellular membranefractions thereof, to a screening system for discovering human SMBPagonists/antagonists characterized by utilizing the transformed cells,the cellular membrane fractions thereof or the isolated recombinanthuman SMBPs, and to human SMBP agonists or antagonists obtainable by thescreening system.

BACKGROUND ART

SM-11044((L)-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluoro-phenyl)propyl] serinepyrrolidine amide hydrobromide)-binding receptor protein (SMBP) wasdiscovered as a new protein that is bound by SM-11044, which is anagonist for β-adrenergic receptors, and by iodocyanopindolol, which isan antagonist for β-adrenergic receptors (Sugasawa, T. et al., J. Biol.Chem. 272, 21244-21252 (1997)). SMBP is a membrane protein resided atlung, ileum, and eosinophil membrane, and is believed to act as areceptor for SM-11044. SM-1T1044 was known to have activities todown-regulate the depolarization-mediated contraction of intestine andto inhibit migration of eosinophils, and has been believed to exert suchSM-111044's functions via SMBP (Sugasawa, T. et al., J. Biol. Chem.,272, 21244-21252 (1997)).

Although the cDNA of a human SMBP was recently cloned (InternationalPublication No. WO 98/26065), it was not reported that SM-11044 binds toany recombinant protein translated from the cDNA. In other words, therehas been no report showing that a human SMBP is expressed at an elevatedlevel to the extent that its ligand-binding activity can be measured,and, therefore, any SMBP has not yet been established in its particularavailability.

DISCLOSURE OF THE INVENTION

The present invention aims to provide a recombinant human SMBPexhibiting ligand-binding activities, and its use. More particularly, itaims to provide transformed cells that are designed to express arecombinant human SMBP at an elevated level to the extent that itsligand-binding activity can be measured by deleting the polythymidinesequence from the base sequence of the 3′-untranslated region, cellularmembrane fractions thereof, and recombinant human SMBPs isolated fromthe transformed cells or the cellular membrane fractions thereof, aswell as a screening system for discovering human SMBPagonists/antagonists characterized by utilizing the transformed cells,the cellular membrane fractions thereofor the isolated recombinant humanSMBPs, and human SMBP agonists or antagonists obtainable by thescreening system.

As mentioned above, SMBP has been believed to be a receptor thatmediates actions to down-regulate the depolarization-mediatedcontraction of intestine and to inhibit migration of eosinophils.Accordingly, it is expected that substances exhibiting an agonisticactivity for SMBP would bind to SMBP to exert the functions as mentionedabove, thereby leading to pharmaceutical compositions for treatinginflammatory diseases involving eosinophil infiltration, asthma, orbowel diseases.

The present inventors attempted to construct a screening system fordiscovering efficiently ligands binding to SMBP, which comprises usingSMBP in view of the development of such pharmaceutical compositions.International Publication No. WO 98/26065 describes that westernblotting with use of anti-human SMBP antibody revealed that a SMBPprotein was expressed by COS cells transformed with the recombinanthuman SMBP cDNA. However, the SMBP protein was expressed in a quitesmall amount, and, consequently, it has no t been reported that anyhuman SMBP is expressed in a sufficiently high level to constructscreening systems. In fact, the inventors obtained the relevant humanSMBP cDNA fragment (SEQ ID NO: 1) from the applicant of theInternational Application (WO 98/26065), Vetigen, and transformed thecDNA into COS-1 cells or CHO-K1 cells. Then, the inventors determined aligand-binding activity of the transfectants, but found no activity. Dueto these facts, the inventors believed either of 1) that any protein hadnot translated from the human SMBP cDNA, or 2) that, even if a proteinwas translated, it had not been expressed at an elevated level to theextent that its ligand-binding activity can be measured.

The present inventors presumed that drawbacks involving the structure ofthe cDNA would cause no or little expression of the protein. Restudy ofthe base sequence of SEQ ID NO: 1 revealed that the 3′-untranslatedregion contains a polythymidine sequence consisting of a consecutivesequence of as many as 37 thymidines, and that the polyuridine sequenceof the mRNA corresponding to the polythymidine sequence binds to thepolyadenine tail (poly-A) residing at the 3′-terminus of the SMBP mRNAto form certain secondary structure, which would thereby restraintranslation into proteins.

On the basis of the above presumption, by deleting the polythymidinesequence from the 3′-untranslated region in the base sequence of thehuman SMBP cDNA depicted in SEQ ID NO: 1, the inventors havesuccessfully expressed a recombinant human SMBP at an elevated level tothe extent that its ligand-binding activity can be measured for thefirst time. Further, the inventors have successfully established, forthe first time, a screening system for discovering SMBPagonists/antagonists that are effective in a human, owing to theavailability of such measurement of ligand-binding activity.

The present invention has been completed on the basis of the findings asdescribed above.

Thus, the present invention relates to:

(1) A process for expressing a recombinant protein at an elevated level,which comprises deleting a sequence comprising the polythymidinesequence from the base sequence of the 3′-untranslated region;

(2) A process for expressing a recombinant human SMBP at an elevatedlevel, which comprises;

(a) preparing a DNA wherein a sequence comprising the polythymidinesequence is deleted from the 3′-untranslated region in the base sequenceof SEQ ID NO: 1;

(b) introducing the DNA of the above (a) into an expression vector;

(c) transforming a host cell with the expression vector of the above(b); and

(d) culturing the transformed cells of the above (c) under anappropriate condition;

(3) A DNA encoding a recombinant human SMBP, which is characterized inthat;

(e) a sequence comprising the polythymidine sequence is deleted from a3′-region from position 1875 in the base sequence of SEQ ID NO: 1; and

(f) the recombinant human SMBP that is a translation product of the DNAcan be expressed at an elevated level to the extent that itsligand-binding activity can be measured;

(4) The DNA of the above (3) wherein a sequence comprising all or partof the base sequence from positions 1899 to 1935 of SEQ ID NO: 1 isdeleted;

(5) The DNA of the above (4) wherein the portion of the base sequencefrom positions 1.875 to 2072 of SEQ ID NO: 1 is deleted.

(6) The DNA of the above (5), which consists of the base sequence of SEQID NO: 3.

(7) An expression vector which carries the DNA of any one of the above(3) to (6).

(8) A transformed cell expressing a recombinant protein at an elevatedlevel to the extent that its ligand-binding activity can be measured,which is obtainable by the process of the above (1) or (2), or acellular membrane fraction thereof.

(9) A transformed cell expressing a recombinant human SMBP at anelevated level to the extent that its ligand-binding activity can bemeasured, which is obtainable by the process of the above (2), or acellular membrane fraction thereof.

(10) The transformed cell of the above (9), which is obtainable byculturing cells transformed with the expression vector of the above (7)under an appropriate condition, or a cellular membrane fraction thereof.

(11) A process for preparing a recombinant human SMBP, which comprisesisolating the recombinant human SMBP from the transformed cells or the;cellular membrane fractions thereof according to the above (9) or (10).

(12) A recombinant human SMBP obtainable by the process of the above(11).

(13) A screening system for discovering a human SMBP agonist orantagonist, which comprises utilizing the transformed cell or thecellular membrane fraction thereof according to the above (9) or (10),or the recombinant human SMBP of the above (12).

(14) A human SMBP agonist or antagonist obtainable by the screeningsystem of the above (13).

(15) A pharmaceutical composition for inhibiting migration ofeosinophils, or for relaxing the contraction of intestine, whichcomprises the agonist of the above (14).

According to the present invention, a recombinant human SMBP has beensuccessfully expressed at an elevated level to the extent that itsligand-binding activity can be measured, by deleting the polythymidinesequence from the 3′-untranslated region of the DNA encoding the humanSMBP. It is understood that this would result from the consequence ofthe right translation into a protein provided by deletion of thepolythymidine sequence that restrains the translation into a protein dueto certain secondary structure formed by binding the polyuridinesequence in the mRNA corresponding to the polythymidine sequence to thepolyadenline tail (poly-A) residing at the 3′-terminus of the mRNA.

Besides the human SMBP DNA as shown above, it is believed that similareffects can be also obtained by deleting a polythymidine sequence incases of DNAs of other recombinant proteins having a polythymidinesequence in the 3′-regions. Accordingly, the present invention providesa process for expressing recombinant proteins in general at elevatedlevels, as well as transformed cells expressing recombinant proteinsobtainable by the process, and the cellular membrane fractions thereof.

Particular steps of the process for conducting the elevated expressionas mentioned above are provided below, taking a recombinant human SMBPfor instance. Thus, the process for expressing a recombinant human SMBPat an elevated level comprises;

(a) preparing a DNA wherein a sequence comprising the polythymidinesequence is deleted from the 3′-untranslated region in the base sequenceof SEQ ID NO: 1;

(b) introducing the DNA of the above (a) into an expression vector;

(c) transforming a host cell with the expression vector of the above(b); and

(d) culturing the transformed cells of the above (c) under anappropriate condition. Details of each step of these (a) to (d) aredescribed hereinafter.

In the present invention, the term “DNA” refers to any DNA as long asthe DNA encodes a recombinant human SMBP, of which the polythymidinesequence is deleted from the 3′-untranslated region in the base sequenceof SEQ ID NO: 1. Specific examples include a DNA encoding a recombinanthuman SMBP, which is characterized in that; (a) a sequence comprisingthe polythymidine sequence is deleted from a 3′ region from position1875 in the base sequence of SEQ ID NO: 1; and, as a consequence of thedeletion, (b) the translation product of the DNA, the recombinant humanSMBP, may be expressed at an elevated level to the extent that itsligand-binding activity can be measured.

In this context, “a DNA encoding human SMBP” may be readily cloned onthe basis of the base sequence of human SMBP described in WO 98/26065 byusing as PCR primers or probes for hybridization an appropriate portionin the base sequence according to conventional methods (consulting astandard text such as “Molecular Cloning”, 2nd ed., Cold Spring HarborLaboratory Press (1989)). Further, alterations such as substitution,deletion, or addition may be also made to the cloned DNA according to“Molecular Cloning” 2nd Edt. Chapter 15, Cold Spring Harbor LaboratoryPress (1989), and such altered SMBPs-encoding DNAs fall within the scopeof the DNA encoding a recombinant human SMBP of the present invention aslong as the expressed products of the DNAs, altered SMBPs, exhibit abinding activity to ligands such as SM-11044.

Among these DNAs encoding recombinant human SMBPs, specific examples ofthe present invention include a DNA which is characterized in that; (a)a sequence comprising the polythymidine sequence is deleted from a 3′region from position 1875 in the base sequence of SEQ ID NO: 1; and (b)the translation product of the DNA, the recombinant human SMBP, may beexpressed at an elevated level to the extent that its ligand-bindingactivity can be measured.

In this connection, with respect to the base sequence up to position1875 in the sequence of human SMBP-DNA of SEQ ID NO: 1, the basesequence up to position 1875 in the sequence of SEQ ID NO: 1, or asequence wherein the sequence contains the above alteration in said basesequence, and the expressed product of the DNA, a recombinant SMBP,exhibits a binding activity to ligands such as SM-11044 are fallenwithin the scope of the present invention. Specific examples include thesequence up to position 1874 in SEQ ID NO: 1, the sequence up toposition 1827 in SEQ ID NO: 3, and the like.

With respect to a 3′ region from position 1875 in the base sequence ofSEQ ID NO: 1, any DNA wherein a sequence comprising “polythymidinesequence” expected to inhibit the expression of a human SMBP protein isdeleted falls within the scope of the present invention. Methods fordeleting a sequence comprising the polythymidine sequence include amethod for the deletion wherein suitable restriction enzyme sitespositioned at each side of the polythymidine are utilized if any, and amethod for the deletion involving well-known techniques such as PCR(Molecular Cloning: A Laboratory Manual 2nd Edt. Chapters 1-3, ColdSpring Harbor Laboratory Press (1989)).

In this connection, the term “polythymidine sequence” refers to asequence comprising consecutive thymidines, of which the deletion leadsto the expression of the translation product, the recombinant humanSMBP, at an elevated level to the extent that its ligand-bindingactivity can be measured.

Examples of the method for measuring a ligand-binding activity includethe method described in J.Biol.Chem., 272, 21244-21252 (1997). Themethod in principle comprises determining a binding reactivity to 1 nM[¹²⁵I]-iodocyanopindolol used as a ligand, determining a nonspecificbinding reactivity of iodocyanopindolol by use of 10⁻⁴ M SM-11044, andsubtracting the nonspecific binding reactivity from the bindingreactivity so as to measure a ligand-binding activity of SMBP protein(J. Biol. Chem., 272, 21244-21252 (1997)).

Specifically, a human SMBP expression vector is prepared by introducinga candidate DNA for the DNA of the present invention into an expressionvector, and a transformed cell is prepared by introducing the SMBPexpression vector into a host cell. Then, the resultant transformedcells or cellular membrane fractions thereof are subjected to the systemfor measuring a ligand-binding activity as shown above (the expressionvector, the transformed cells, and the cellular membrane fractionsthereof are further described hereinafter). Examples of the method formeasuring a ligand-binding activity include the substantially samemethod as that described in J. Biol. Chem., 272, 21244-21252 (1997)mentioned above, and a method that is detailed in Example 6.Specifically, a 96-well Multiscreen plate (Millipore) in which a pieceof glass fiber paper is placed on the bottom of the wells is treatedwith Tris-HCl buffered saline containing 0.3% polyethyleneimine (Sigma)(reconstituted to pH7.4 with 6N HCl), and washed by vacuum filtrationwith Tris-HCl buffered saline (pretreatment). Then, 200 μl of Tris-HClbuffered saline containing 1 nM [¹²⁵I]-iodocyanopindolol (Amersham) anda cellular membrane fraction as mentioned above (50 μg of membraneprotein) that have been incubated at 37° C. for 30 minutes is added toeach well on the 96-well Multiscreen plate, and are washed by vacuumfiltration. The cellular membrane fraction is harvested on the glassfiber paper, and washed by vacuum filtration with 200 μl of anice-cooled Tris-HCl buffered saline. Then, the amount of[¹²⁵I]-iodocyanopindolol bound to the membrane fraction trapped on thepaper is determined by a gamma counter to represent a total binding.Nonspecific binding of [¹²⁵I]-iodocyanopindolol is determined byconducting an incubation as mentioned above in the presence of 10⁻⁴ MSM-11044 (Sumitomo Pharmaceuticals Co., Ltd. it can be preparedaccording to the process described in Japanese Patent Publication(kokai.) No. 132935/1985, Japanese Patent Publication (kokoku) No.50499/1993) and then conducting similar procedures to those mentionedabove. A ligand-binding to SMBP may be calculated by subtracting thenonspecific binding from the total binding.

DNAs of the present invention can be readily selected by subjectingtransformed cells introduced with a candidate DNA for the DNA of thepresent invention, or cellular membrane fractions thereof to the systemfor measuring a ligand-binding activity as shown above. The system forthe measurement may be appropriately modified as far as the commonknowledge of those skilled in the art. For example, SM-11044, BRL-35135A(Smith Kline Beecham), or alprenolol (Ciba Geigy) labeled with [¹²⁵I] or[³H] may be used instead of [¹²⁵I]-iodocyanopindolol.

Among the DNAs of the present invention, suitable examples include a DNAwherein a sequence comprising all or part of the polythymidine sequenceresiding from positions 1899 to 1935 is deleted from the 3′-untranslatedregion in the base sequence of SEQ ID NO: 1. In this connection, “thepart” is preferably about 30 bases in length since it should have beenbound to the polyadenine tail (poly-A) attached to the 3′-terminus ofthe SMBP mRNA to form certain secondary structure, although any lengthin the part may be acceptable as long as a recombinant human SMBP can beexpressed at an elevated level to the extent that its ligand-bindingactivity can be measured.

More suitable examples of the DNA of the present invention includes aDNA wherein the sequence from positions 1875 to 2072 is deleted from thebase sequence of SEQ ID NO: 1, and even more suitable examples includethe human SMBP DNA consisting of the base sequence of SEQ ID NO: 3.

The DNA of the present invention as mentioned above may be incorporatedinto an expression vector according to conventional methods to obtain aSMBP expression vector carrying the DNA of the invention.

In this connection, the expression vectors to be incorporated with theDNA of the present invention may be any vector capable of expressingefficiently then subject in a host cell, and preferably include apcDNA3.1 derivative, pRc/RSV, pRc/CMV, a pEF derivative (all of them arefrom Invitrogen), pIRESneo (Clontech), and a pREP9 derivative(Invitrogen).

The SMBP expression vector thus prepared may be transformed into hostcells to prepare transformed cells wherein the expression vector of thepresent invention is retained stably in chromosomes of the host cells.In this connection, host cells may be any cell as long as a foreign genemay be stably integrated into chromosomes of the host cells, and mammalcells are preferred. Examples of the host cells include CHO cells, L929cells, C127 cells, and BALB/c3T3 cells as well as variants thereofwherein a dihydrofolate reductase or thymidine kinase function isdefective.

Examples of methods for transforming the expression vector of thepresent invention into host cells include the calcium phosphate method(J. Virol.,52, 456-467 (1973)), a method involving LT-1 (Panvera), and amethod involving lipids for gene-introduction (Lipofectamine,Lipofectin; Gibco-BRL). After the transformation, the cells may becultured in a conventional medium containing a selective marker (forexample, Zeocin in the case of using pcDNA3.1/Zeo(+) shown above as anexpression vector) to select the transformed cells wherein theexpression vector of the present invention is stably retained inchromosomes of the host cells.

The transformed cells thus obtained may be continuously cultured underan appropriate condition to prepare transformed cells of the presentinvention wherein a recombinant human SMBP is expressed on the cellularmembrane at an elevated level to the extent that its ligand-bindingactivity can be measured.

The term “appropriate condition” refers to a condition wherein acultivation is conducted at 37° C. under 5% CO₂ in a culture mediumsuitable for respective host cell, and examples include a conditionwherein CHO cells are cultured at 37° C. under 5% CO₂ in HAM'S F-12medium containing 10% bovine calf serum.

Cellular membrane fractions of the present invention can be preparedfrom the transformed cells thus obtained wherein a human SMBP isexpressed at an elevated level. For example, a process for preparing thecellular membrane fractions may comprise adding to the cells a hypotonichomogenate buffer (10 mM Tris-HCl buffer, 1 mM EDTA, 0.5 mM PMSF or 1 mMAEBSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin, pH7.4), allowing themixture to stand at 4° C. for about 30 minutes so as to destroy thecells due to the hypotonic condition, homogenizing it by the pipetting,and centrifuging the homogenate at 4° C. at 50000×g for about 30minutes, thereby obtaining the cellular membrane fractions of thepresent invention.

Alternatively, for example, the method described by F. Pietri-Rouxel etal. (Eur. J. Biochem., 247, 1174-1179 (1997)) may be used to prepare thecellular membrane fractions of the present invention.

A recombinant human SMBP of the present invention can be isolated fromthe transformed cells of the cellular membrane fractions of the presentinvention thereof thus obtained. Specifically, for example, the methodof R. G. Shorr, et al. (Proc. Natl. Acad. Sci. USA, 79, 2778-2782(1982); J. Biol. Chem. 257, 12341-12350 (1982)) may be used to obtain acrude extract of a human SMBP of the present invention. Further, amethod for purifying a human SMBP from the crude extract is exemplifiedby the method of J. L. Benovic, et al. (Biochem., 23, 4510-4518 (1984)).

Specific examples of the recombinant human SMBP of the present inventioninclude a human SMBP consisting of the amino acid of SEQ ID NO: 2 or SEQID NO: 4, and a altered SMBP consisting of said amino acid that containssubstitution, deletion, and/or addition is also fallen within the scopeof the recombinant human SMBPs of the present invention as long as thelatter has a binding activity to ligands such as SM-11044.

Agonists or antagonists binding to the human SMBP can be screened by useof either of the transformed cells expressing the recombinant human SMBPat an elevated level, the cellular membrane fractions thereof, or theisolated recombinant human SMBP, each of which are as obtained, above.

The screening by use of the cellular membrane fractions of thetransformed cells expressing the recombinant human SMBP at an elevatedlevel may be conducted for example by the following method.

First of all, a Tris-HCl buffered saline containing the cellularmembrane fraction of the present invention (50-200 μg membrane protein)and 1 nM [¹²⁵I]-iodocyanopindolol is incubated at 37° C. for 30 minutes,and the reaction is added to each well on a 96-well Multiscreen platethat has been treated by a similar pretreatment to that in “the methodfor measuring a ligand-binding activity” as mentioned above, then beingaspirated by vacuum filtration. Subsequently, a similar treatment tothat in “the method for measuring a ligand-binding activity” asmentioned above is conducted, and the amount of [¹²⁵I]-iodocyanopindololbound to the membrane fraction trapped on the paper is determined by agamma counter to give a binding, which represents binding A. Then, theincubation as shown above is conducted in the presence of a testcompound at a normal range of concentrations (10⁻¹²-10⁻⁴M), and then asimilar procedure is conducted to give a binding, which representsbinding B. A binding that is provided by use of 10⁻⁴M SM-11044 insteadof a test compound represents binding C. Accordingly, when the valuesubtracted binding B from binding A is equivalent to one subtractedbinding C from binding A, the test compound is estimated to have 100%SMBP-binding activity, and, when the value is the half, the compound isestimated to have the 50%. SMBP ligands thus selected are subjected toan assay as described in either J. Biol. Chem., 272, 21244-21252 (1997),Eur. J. Pharmacol. 216, 207-215 (1992), or Agents Actions 37, 233-237(1992). At that time, when down-regulating the contraction of intestineor inhibiting the migration of eosinophils equivalently to or more thanSM-11044, the ligands may be a SMBP agonist, whereas when showing theinverse activities, they may be a SMBP antagonist.

The screening by use of the transformed cells expressing the human SMBPof the present invention at an elevated level may be conducted forexample by the following method.

First of all, the transformed cells expressing the human SMBP of thepresent invention at an elevated level that have been washed withDulbecco's phosphate buffered saline (Gibco) are incubated with 1-5 mMEDTA-Dulbecco's phosphate buffered saline at room temperature, and thenremoved from the culture dish. After centrifuged (400×g, 10 min 4° C.)and the supernatant being removed by aspiration, the cells are suspendedby the pipetting in Tris-HCl buffered saline containing 0.2% bovineserum albumin. The cells at 5×10⁵, 1 nM [¹²⁵I]-iodocyanopindolol, andTris-HCl buffered saline containing 0.2% bovine serum albumin areincubated at 37° C. for 30 minutes, and the reaction is added to eachwell on a 96 well-Multiscreen plate that has been treated by a similarpretreatment to that in “the method for measuring a ligand-bindingactivity”, as mentioned above, the being aspirated by vacuum filtration.Subsequently, a similar treatment to that that in “the method formeasuring a ligand-binding activity” as mentioned above is conducted,and the amount of [¹²⁵I] iodocyanopindolol bound to the membranefraction trapped on the paper is determined by a gamma counter to give abinding, which represents binding A. Then, incubation as shown above isconducted in the presence of a test compound at a normal rage ofconcentrations (10⁻¹²-10⁻⁴M), and a similar procedure is conducted togive a binding, which represents binding B. A binding that is providedby use of 10⁻⁴M SM-11044 instead of a test compound represents bindingC. Subsequent procedures for the estimation are as shown above.

The screening by use of the recombinant human SMBP isolated from thetransformed cells or the cellular membrane fractions thereof accordingto the present invention may be conducted for example by the followingmethod.

First of all, an isolated SMBP standard is prepared by solubilizing thetransformed cells or the cellular membrane fractions thereof by use of asuitable solubilizer, preferably, β-D-octylglucoside. Two hundreds fiftyμl of Tris-HCl buffered saline containing the isolated SMBP standard and1 nM [¹²⁵I]-iodocyanopindolol is incubated at 37° C. for 30 minutes, andthen 250 μl of an ice-cooled Tris-HCl buffered saline is added thereto,thereby quenching the reaction. The reaction is applied to Sephadex G-50column (Pharmacia) that have been equilibrated with Tris-HCl bufferedsaline, and eluted with Tris-HCl buffered saline. The amount of[¹²⁵I]-iodocyanopindolol bound to the SMBP fraction thus isolated by thegel filtration is determined by a gamma counter to give a binding, whichrepresents binding A. Subsequently, incubation as shown above isconducted in the presence of a test compound at a normal rage ofconcentrations (10⁻¹²-10⁻⁴M), and a similar procedure is conducted togive a binding, which represents binding B. A binding that is providedby use of 10⁻⁴M SM-11044 instead of a test compound represents bindingC. Subsequent procedures for the estimation are as shown above.

The screening systems as described above can be modified as appropriatewithin the knowledge of those skilled in the art.

Human SMBP agonists obtainable by the above screening systems are usefulfor an agent for inhibiting migration of eosinophils, or relaxing thecontraction of intestine, and, specifically, are useful as a medicamentor prophylactic for inflammatory diseases, asthma, or bowel diseasessuch as allergic gastrointestinal symptoms, involving eosinophilinfiltration.

Agonists or antagonists obtainable by the screening systems of thepresent invention may be used for a pharmaceutical composition as shownabove in accordance with conventional manners. For example, suchsubstances can be administered orally in the form employed commonly inthe art, such as tablets, capsules, syrups, suspensions, or the like.Such substances can also be administered parenterally in the form of,for example, solutions, emulsions, suspensions, or the like, as well ascan be administered by rectal route in the form of suppositories. Suchsuitable dosage forms may be prepared in accordance with conventionalmanners by combining the active ingredient with conventional carriers,excipients, binders, stabilizing agents, or the like. Injectableformulations can additionally contain buffers, solubilizing agents, orisotonizating agents. Doses and frequencies vary depending on, forexample, the disease and condition to be treated, the age and weight ofa particular patient, a dosage form, and the like, and a typical dailydose for adults of active ingredients in case of oral formulation's mayrange about 1 mg to about 1000 mg, preferably, about 10 to about 500 mg,which may be administered at a time or in portions. In case of theinjectable formulations, a dose of active ingredients may range about0.1 mg to about 500 mg, preferably, about 3 mg to about 100 mg, whichmay be administered at a time or in portions.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the values of the ligand-binding activitiesthat were measured using the cellular membranes of COS-7 cellstransfected with pcDNA3.1/Zeo(+), SMBP-pcDNA3.1/Zeo(+),SMBP-Kozak-pcDNA3.1/Zeo(+), or SMBP-Kozak-pcDNA3.1/Zeo(+) poly-T free,wherein these are indicated by CHO-pcDNA3.1, CHO-SMBP, CHO-SMBP-Kozak,and CHO-SMBP-Kozak-poly T free, respectively. The results representmeans±standard error of the duplicate experiments. The axis of ordinatesindicates the amounts (CPM) of [¹²⁵I]-iodocyanopindolol boundspecifically to the SMBP. The symbols “**”, “+”, and “N.S.” mean thatthere is a significant difference (p<0.01) in relation to CHO-pcDNA3.1,that there is a significant difference (p<0.05) in relation to CHO-SMBP,and that there is no significant difference in relation to ECHO-pcDNA3.1or CHO-SMBP, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is further illustrated by the following examples,but is not restricted by these examples in any way.

EXAMPLE 1 Introduction of BamHI/XbaI-cleaved Fragment of SMBP-cDNA IntoExpression Vector

The BamHI/XbaI cleaved fragment of SMBP-cDNA described in WO 98/26065was obtained from the applicant of the patent application, Vetigen. Thebase sequence of the cDNA was sequenced by Takara Shuzo Co., Ltd. inaccordance with a conventional method. The base sequence determined isshown in SEQ ID NO: 1, and the amino acid sequence of the proteinencoded by the cDNA is shown in SEQ ID NO: 2. The base sequence of SEQID NO: 1 contains the base sequence disclosed as SEQ ID NO: 13 in WO98/26065, and is further flanked by both 5′ and 3′ sequences.

Subsequently, the cDNA fragment was introduced into an expressionvector, pcDNA3.1/Zeo(+) (Invitrogen), which had been cleaved with BamHIand XbaI, to give an expression plasmid, SMBP-pcDNA3.1/Zeo(+). E. coliJM 109 (Toyobo Co. Ltd.) was transformed with the plasmid SMBPpcDNA3.1/Zeo(+), and cultured overnight on a LB plate containing 50μg/ml ampicillin to give transformants. Plasmids prepared from thetransformants according to a conventional procedure were cleaved withBamHI and XbaI to confirm that the SMBP-cDNA (about 2 kb) was insertedinto the fragment. Further, cleavage with ApaI confirmed the orientationof the inserted sequence.

EXAMPLE 2 Binding of Kozak Sequence to SMBP-cDNA Fragment andIntroduction Into Expression Vector

In surrounding sequences of ATG, a start codon, of the SMBP-cDNAfragment (at positions 49-51 in SEQ ID NO: 1), the Kozak's consensussequence (ACCATGG SEQ ID NO:5) presumed to be necessary to translateefficiently mRNAs into proteins is not found. Accordingly, thesurrounding sequence of the start codon of SMBP-cDNA fragment wasreplaced with ACCATGG (SEQ ID NO:5) as shown below in order to improveexpression efficiency of proteins.

First, the following, adapter containing the Kozak's consensus sequenceand HindIII/NotI lcleavage sites was prepared by annealing two types ofdifferent oligomers at 70° C. for 10 minutes:

Adapters Containing, the Kozak's Sequence

5′-AGC TTC CAC CAT GGC-3′ (SEQ ID NO:6)

3′-AG GTG GTA CCG CCG G-5′ (SEQ ID NO:7)

The adapter thus prepared was incorporated into the expression vectorpcDNA3.1/Zeo(+), which had been cleaved with HindIII/NotI to giveaplasmid, Kozak-pcDNA3.1/Zeo(+). E. coli JM 109 was transformed with theplasmid Kozak-pcDNA3.1 /Zeo(+), and cultured overnight on a LB platecontaining 50 μg/ml ampicillin to give transformants. The multicloningsites in pcDNA3.1/Zeo(+) contain a BamHI cleavage site between theHindIII-NotI sites, and, therefore, any plasmid without the insertedadapters would be cleaved with BamHI. Thus, the presence of the insertedadapters was determined by whether or not plasmids prepared from thetransformants could be cleaved with BamHI.

Subsequently, the SMBP-cDNA fragment prepared by digesting theBamHI/XbaI-cleaved fragment of the SMBP-cDNA with NotI was introducedinto Kozak-pcDNA3.1/Zeo(+), which had been cleaved with NotI, to give anexpression plasmid, SMBP-Kozak-pcDNA3.1/Zeo(+). E. coli JM 109 (ToyoboCo. Ltd.) was transformed with the plasmid SMBP-Kozak-pcDNA3.1 /Zeo(+),and cultured overnight on a LB plate containing 50 μg/ml ampicillin togive transformants. The plasmids prepared from the transformants werecleaved with HindIII and XbaI to confirm that the SMBP-cDNA (about 2 kb)was inserted into the plasmid. Further, cleavage with ApaI confirmed theorientation of the inserted sequence.

The amino acid sequence of the SMBP protein translated from the plasmidSMBP-Kozak-pcDNA3.1/Zeo(+) prepared as shown above was the sequencewherein the three amino acids at positions 2 to 4, His-Ala-Arg, isdeleted from the amino acid sequence of SEQ ID NO: 2.

EXAMPLE 3 Deletion of Polythymidine Sequence from Expression PlasmidSMBP-Kozak-pcDNA3.1 /Zeo(+): and Reintroduction into Expression VectorpcDNA3.1 /Zeo(+)

A polythymidine sequence consisting of a consecutive sequence of 37thymidines (poly-T) exists downstream of the stop codon (TAG) in theSMBP-cDNA, which 'sequence is between positions 1899-1935 in the basesequence of SEQ ID NO: 1. The inventors presumed that the poly-U in themRNA corresponding to the poly-T should bind to the polyadenine tail(poly-A) residing at the 3′-terminus of the SMBP-mRNA to form certainsecondary structure, which would destabilize the m-RNA, or block itstransport through the nuclear membrane, thereby restraining translationinto proteins. Thus, the base sequence downstream of position 1875 wasexcised from the base sequence of SEQ ID NO: 1 using restriction enzymeKsp6321 to give a SMBP-cDNA fragment wherein the portion of 198 basescontaining the poly-T part was deleted, and the fragment was thenintroduced into an expression vector in accordance with the proceduresas shown below.

Specifically, the expression plasmid SMBP-Kozak-pcDNA3.1 /Zeo(+) asprepared in Example 2 was cleaved with Ksp632I to isolate a fragment(about 2.9 kb) wherein a SMBP-cDNA from which the portion of 198 basescontaining the poly-T part was deleted, and a portion of pcDNA3.1/Zeo(+)were bound together. Then, the fragment was cleaved with HindIII, and aHindIII/Ksp632I-cleaved fragment of the SMBP-cDNA (about 1.8 kb) wasisolated. The fragment was blunted with T4DNA polymerase (Takara Syuzo),and the blunt-ended fragment was introduced into an expression vectorpcDNA3.1/Zeo(+) cleaved with EcoRV to give an expression plasmidSMBP-Kozak-pcDNA3.1 /Zeo(+) poly-T free. The base sequence of theSMBP-cDNA carried on the expression plasmid SMBP-Kozak-pcDNA3.1 /Zeo(+)poly-T free is shown in SEQ ID NO: 3, and the amino acid sequencethereof is shown in SEQ ID NO: 4.

E. coli JM 109. (Toyobo Co. Ltd.) was transformed with the plasmidSMBP-Kozak-pcDNA3.1/Zeo(+) poly-T free, and cultured overnight on a LBplate;containing 50 μg/ml ampicillin to give transformants. The plasmidsprepared from the transformants were cleaved with HindIII/XbaI toconfirm that the SMBP-cDNA (about 2 kb) was inserted into the plasmid.Further, cleavage with ApaI confirmed the orientation of the insertedsequence.

EXAMPLE 4 Introduction of Human SMBP-expression Plasmid Into AnimalCells

CHO-k1 cells were plated into wells of a 6-well plate at 2×10⁵cells/well, and cultured for 24 hours in HAM's F-12 medium containing10% fetal calf serum. The cells were transformed with pcDNA3.1 /Zeo(+)(control);, or each of human SMBP protein-expression plasmids,SMBP-pcDNA3.1/Zeo(+), SMBP-Kozak-pcDNA3.l1/Zeo(+) andSMBP-Kozak-pcDNA3.1/Zeo(+) poly-T free as prepared in Examples 1 to 3,using lipids for gene incorporation (Lipofectamine; Gibco-BRL). From thefifth day after the transformations, the cells were cultured in HAM'sF-12 medium containing 1.0 mg/ml Zeocin (Invitrogen) and 10% fetal calfserum, and cells wherein the plasmids were integrated in the chromosomeswere selected.

EXAMPLE 5 Preparation of CHO Cellular Membrane Fractions ContainingHuman SMBP Protein

CHO cells containing the human SMBP protein obtained in Example 4 werecultured in an adherent manner in a culture dish having a diameter of 10cm, and washed with phosphate buffered saline (PBS). To the cells, 5 mlof an ice-cooled, hypotonic homogenate buffer (10 mM Tris-HCl buffer, 1mM EDTA, 0.5 mM PMSF or 1 mM AEBSF, 5 μg/ml aprotinin, 5 μg/mlleupeptin, pH7.4) was added, and the mixture was allowed to stand at 4°C. for 30 minutes, thereby destroying the cells due to the hypotoniccondition. The destroyed material was homogenized by the pipetting, andthe homogenate was centrifuged at 50000×g at 4° C. for about 30 minutesto give sediment of crude membrane fractions. The sediment was suspendedin Tris-HCl buffered saline (Tris-HCl buffer, 154 mM sodium chloride, pH7.4), and the suspension was stored at −80° C. Each time used, a portionof this was thawed. Protein concentration was determined by a ProteinAssay Kit (Bio-Rad) wherein bovine serum albumin is used as a standard.

EXAMPLE 6 Measurement of Lizand-binding Activity of Human SMBP Protein

Sugasawa et al. (Sugasawa, T. et al., J. Biol. Chem., 272, 21244-21252(1997)) reported that aligand-binding reactivity of a human SMBP proteincan, be measured by use of 1 nM [¹²⁵I]-iodocyanopindolol (2000 Ci mmol;Amersham) as a ligand, and, specifically, the ligand-binding reactivityof a human SMBP protein can be estimated by determining a nonspecificbinding reactivity of iodocyanopindolol by use of 10⁻⁴M SM-11044, andsubtracting the non-specific binding reactivity from the bindingreactivity. According to the instructions of this literature, aligand-binding activity of a SMBP protein was measured. Further, thebinding assay was conducted using a 96-well microtiter plate in order toaccelerate the assay.

First, a 96-well Multiscreen plate (Millipore) in which a piece of glassfiber paper was placed on the bottom of the wells was supplemented withTris-HCl buffered saline containing 0.3% polyethyleneimine (Sigma)(reconstituted to pH7.4 with 6N HCl), treated for 30 minutes or more,and washed aspirating with Tris-HCl buffered saline (pretreatment) byvacuum filtration.

Subsequently, 200 μl of Tris-HCl buffered saline containing 1 nM[¹²⁵I]-iodocyanopindolol and each cellular membrane fraction as preparedin Example 5 (50 μg of membrane protein) that had been incubated at 37°C. for 30 minutes was added to each well on the 96-well Multiscreenplate, and were aspirated by vacuum filtration. The cellular membranefractions were harvested on the glass fiber paper, and washed four timesby vacuum filtration with 200 μl of an ice-cooled Tris-HCl bufferedsaline. The amounts of [¹²⁵I]-iodocyanopindolol bound to the membranefraction trapped on the paper were determined by a gamma counter torepresent total bindings. Nonspecific bindings of[¹²⁵I]-iodocyanopindolol were determined by conducting incubations asmentioned above in the presence of 10⁻⁴ M SM-11044, and then conductingsimilar procedures to those mentioned above. In each case, thenonspecific binding was subtracted from the total binding to give aligand-binding to SMBP (a specific binding). The results are shown inFIG. 1.

The membrane fraction of the CHO cells that were transformed with theexpression plasmid introduced with the SMBP-cDNA described in WO98/26065 (Example 1) (CHO-SMBP in FIG. 1), and the membrane fraction ofthe| CHO cells that were transformed with the expression plasmidintroduced with the Kozak sequence (Example 2) (CHO-SMBP-Kozak inFIG. 1) did not show any significant ligand-binding activities comparedto the control (CHO-pcDNA3.1 in FIG. 1). Contrarily, the membranefraction of the CHO cells that were transformed with the expressionplasmid wherein the Kodak sequence was introduced, and the poly-Tsequence was deleted (Example 3) (CHO-SMBP-Kozak-poly T free in FIG. 1)did show a significant ligand-binding activity compared to the controland to the CHO-SMBP cells before the deletion of the poly-T sequence.These results demonstrate that the deletion of the poly-T sequencecauses an expression of a SMBP protein on the cellular membranesufficient to show its ligand-binding activity.

EXAMPLE 7 Screening for Ligands of Human SMBP Protein

Two hundreds μl of Tris-HCl buffered saline containing 50 μg of membraneprotein of the cellular membrane fraction of the CHO cells transformedwith SMBP-Kozak-pcDNA3.1/Zeo(+) poly-T free (as prepared in Example 5)and 1 nM [¹²⁵¹I]-iodocyanopindolol is incubated at 37° C. for 30minutes, and the reaction is added to each well on a 96-well Multiscreenplate that has been treated by a similar pretreatment to that in Example6, and aspirated by vacuum filtration. Then, a similar treatment to thatin Example 6 is conducted, and the amount of [¹²⁵I]-iodocyanopindololbound to the membrane fraction trapped on the paper is determined by agamma counter to give a binding, which represents binding A.Subsequently, the incubation as shown above is conducted in the presenceof a test compound at a normal range of concentrations (10⁻¹²-10⁻⁴M),and then a similar procedure is conducted to give a binding, whichrepresents binding B. A binding that is provided by use of 10⁻⁴MSM-11044 instead of a test compound represents binding C. When the valuesubtracted binding B from binding A is equivalent to one subtractedbinding C from binding A, the test compound is estimated to have 100%SMBP-binding activity, and, when the value is the half, the compound isestimated to have the 50%. SMBP ligands thus selected are subjected toan assay as described in either J. Biol. Chem., 272, 21244-21252 (1997),Eur. J. Pharmacol. 216, 207-215 (1992), or Agents Actions 37, 233-237(1992). That procedure makes it possible to determine if the ligandshave a SMBP-agonist activity, i.e., if the ligands down-regulate thecontraction of intestine or if they inhibit the migration ofeosinophils.

INDUSTRIAL APPLICABILITY

Cells that are transformed with an expression plasmid carrying a humanSMBP cDNA wherein the polythymidine sequence is deleted according to thepresent invention can express a human SMBP to the extent that itsligand-binding activity can be measured. Accordingly, transformed cellscontaining a recombinant human SMBP, cellular membrane fractionsthereof, or recombinant human SMBP isolated from said transformed cellsor said cellular membrane fractions according to the present inventioncan be used to screen for ligands binding to a human SMBP. The screeningsystems of the present invention enable to efficiently discover humanSMBP agonists or antagonists. Human tissues might be used in thescreening system for discovering compounds binding to human SMBPs.However, when using human tissues, it is difficult to differentiate theintended ligands from those ligands to be bound to receptors other thanthe SMBPs existing at human tissues, such as human β1-, β2-, orβ3-adrenergic receptors. The screening systems of the present inventionmake it possible to surely and efficiently select human SMBP agonists orantagonists, and, therefore, provides speedy development ofpharmaceutical products such as pharmaceutical compositions for treatingor preventing inflammatory diseases involving migration of eosinophils,antiasthmatic compositions, and pharmaceutical compositions for treatingbowel diseases.

7 1 2072 DNA Homo sapiens CDS (49)..(1794) 1 ggatccacta gtaacggccgccagtgtgct ggaattctgc agatctag atg cat gct 57 Met His Ala 1 cga gcg gccgcc gcg ctg tgg ctg ctg ctg ctg ctg ctg ccc cgg acc 105 Arg Ala Ala AlaAla Leu Trp Leu Leu Leu Leu Leu Leu Pro Arg Thr 5 10 15 cgg gcg gac gagcac gaa cac acg tat caa gat aaa gag gaa gtt gtc 153 Arg Ala Asp Glu HisGlu His Thr Tyr Gln Asp Lys Glu Glu Val Val 20 25 30 35 tta tgg atg aatact gtt ggg ccc tac cat aat cgt caa gaa aca tat 201 Leu Trp Met Asn ThrVal Gly Pro Tyr His Asn Arg Gln Glu Thr Tyr 40 45 50 aag tac ttt tca cttcca ttc tgt gtg ggg tca aaa aaa agt atc agt 249 Lys Tyr Phe Ser Leu ProPhe Cys Val Gly Ser Lys Lys Ser Ile Ser 55 60 65 cat tac cat gaa act ctggga gaa gca ctt caa ggg gtt gaa ttg gaa 297 His Tyr His Glu Thr Leu GlyGlu Ala Leu Gln Gly Val Glu Leu Glu 70 75 80 ttt agt ggt ctg gat att aaattt aaa gat gat gtg atg cca gcc act 345 Phe Ser Gly Leu Asp Ile Lys PheLys Asp Asp Val Met Pro Ala Thr 85 90 95 tac tgt gaa att gat tta gat aaagaa aag aga gat gca ttt gta tat 393 Tyr Cys Glu Ile Asp Leu Asp Lys GluLys Arg Asp Ala Phe Val Tyr 100 105 110 115 gcc ata aaa aat cat tac tggtac cag atg tac ata gat gat tta cca 441 Ala Ile Lys Asn His Tyr Trp TyrGln Met Tyr Ile Asp Asp Leu Pro 120 125 130 ata tgg ggt att gtt ggt gaggct gat gaa aat gga gaa gat tac tat 489 Ile Trp Gly Ile Val Gly Glu AlaAsp Glu Asn Gly Glu Asp Tyr Tyr 135 140 145 ctt tgg acc tat aaa aaa cttgaa ata ggt ttt aat gga aat cga att 537 Leu Trp Thr Tyr Lys Lys Leu GluIle Gly Phe Asn Gly Asn Arg Ile 150 155 160 gtt gat gtt aat cta act agtgaa gga aag gtg aaa ctg gtt cca aat 585 Val Asp Val Asn Leu Thr Ser GluGly Lys Val Lys Leu Val Pro Asn 165 170 175 act aaa atc cag atg tca tattca gta aaa tgg aaa aag tca gat gtg 633 Thr Lys Ile Gln Met Ser Tyr SerVal Lys Trp Lys Lys Ser Asp Val 180 185 190 195 aaa ttt gaa gat cga tttgac aaa tat ctt gat ccg tcc ttt ttt caa 681 Lys Phe Glu Asp Arg Phe AspLys Tyr Leu Asp Pro Ser Phe Phe Gln 200 205 210 cat cgg att cat tgg ttttca att ttc aac tcc ttc atg atg gtg atc 729 His Arg Ile His Trp Phe SerIle Phe Asn Ser Phe Met Met Val Ile 215 220 225 ttc ttg gtg ggc tta gtttca atg att tta atg aga aca tta aga aaa 777 Phe Leu Val Gly Leu Val SerMet Ile Leu Met Arg Thr Leu Arg Lys 230 235 240 gat tat gct cgg tac agtaaa gag gaa gaa atg gat gat atg gat aga 825 Asp Tyr Ala Arg Tyr Ser LysGlu Glu Glu Met Asp Asp Met Asp Arg 245 250 255 gac cta gga gat gaa tatgga tgg aaa cag gtg cat gga gat gta ttt 873 Asp Leu Gly Asp Glu Tyr GlyTrp Lys Gln Val His Gly Asp Val Phe 260 265 270 275 aga cca tca agt caccca ctg ata ttt tcc tct ctg att ggt tct gga 921 Arg Pro Ser Ser His ProLeu Ile Phe Ser Ser Leu Ile Gly Ser Gly 280 285 290 tgt cag ata ttt gctgtg tct ctc atc gtt att att gtt gca atg ata 969 Cys Gln Ile Phe Ala ValSer Leu Ile Val Ile Ile Val Ala Met Ile 295 300 305 gaa gat tta tat actgag agg gga tca atg ctc agt aca gcc ata ttt 1017 Glu Asp Leu Tyr Thr GluArg Gly Ser Met Leu Ser Thr Ala Ile Phe 310 315 320 gtc tat gct gct acgtct cca gtg aat ggt tat ttt gga gga agt ctg 1065 Val Tyr Ala Ala Thr SerPro Val Asn Gly Tyr Phe Gly Gly Ser Leu 325 330 335 tat gct aga caa ggagga agg aga tgg ata aag cag atg ttt att ggg 1113 Tyr Ala Arg Gln Gly GlyArg Arg Trp Ile Lys Gln Met Phe Ile Gly 340 345 350 355 gca ttc ctt atccca gct atg gtg tgt ggc act gcc ttc ttc atc aat 1161 Ala Phe Leu Ile ProAla Met Val Cys Gly Thr Ala Phe Phe Ile Asn 360 365 370 ttc ata gcc atttat tac cat gct tca aga gcc att cct ttt gga aca 1209 Phe Ile Ala Ile TyrTyr His Ala Ser Arg Ala Ile Pro Phe Gly Thr 375 380 385 atg gtg gcc gtttgt tgc atc tgt ttt ttt gtt att ctt cct cta aat 1257 Met Val Ala Val CysCys Ile Cys Phe Phe Val Ile Leu Pro Leu Asn 390 395 400 ctt gtt ggt acaata ctt ggc cga aat ctg tca ggt cag ccc aac ttt 1305 Leu Val Gly Thr IleLeu Gly Arg Asn Leu Ser Gly Gln Pro Asn Phe 405 410 415 cct tgt cgt gtcaat gct gtg cct cgt cct ata ccg gag aaa aaa tgg 1353 Pro Cys Arg Val AsnAla Val Pro Arg Pro Ile Pro Glu Lys Lys Trp 420 425 430 435 ttc atg gagcct gcg gtt att gtt tgc ctg ggt gga att tta cct ttt 1401 Phe Met Glu ProAla Val Ile Val Cys Leu Gly Gly Ile Leu Pro Phe 440 445 450 ggt tca atcttt att gaa atg tat ttc atc ttc acg tct ttc tgg gca 1449 Gly Ser Ile PheIle Glu Met Tyr Phe Ile Phe Thr Ser Phe Trp Ala 455 460 465 tat aag atctat tat gtc tat ggc ttc atg atg ctg gtg ctg gtt atc 1497 Tyr Lys Ile TyrTyr Val Tyr Gly Phe Met Met Leu Val Leu Val Ile 470 475 480 ctg tgc attgtg act gtc tgt gtg act att gtg tgc aca tat ttt cta 1545 Leu Cys Ile ValThr Val Cys Val Thr Ile Val Cys Thr Tyr Phe Leu 485 490 495 cta aat gcagaa gat tac cgg tgg caa tgg aca agt ttt ctc tct gct 1593 Leu Asn Ala GluAsp Tyr Arg Trp Gln Trp Thr Ser Phe Leu Ser Ala 500 505 510 515 gca tcaact gca atc tat gtt tac atg tat tcc ttt tac tac tat ttt 1641 Ala Ser ThrAla Ile Tyr Val Tyr Met Tyr Ser Phe Tyr Tyr Tyr Phe 520 525 530 ttc aaaaca aag atg tat ggc tta ttt caa aca tca ttt tac ttt gga 1689 Phe Lys ThrLys Met Tyr Gly Leu Phe Gln Thr Ser Phe Tyr Phe Gly 535 540 545 tat atggcg gta ttt agc aca gcc ttg ggg ata atg tgt gga gcg att 1737 Tyr Met AlaVal Phe Ser Thr Ala Leu Gly Ile Met Cys Gly Ala Ile 550 555 560 ggt tacatg gga aca agt gcc ttt gtc cga aaa atc tat act aat gtg 1785 Gly Tyr MetGly Thr Ser Ala Phe Val Arg Lys Ile Tyr Thr Asn Val 565 570 575 aaa attgac tagagaccca agaaaacctg gaactttgga tcaatttctt 1834 Lys Ile Asp 580tttcataggg gtggaacttg cacagcaaaa acaaacaaac gcaagaagag atttgggctt 1894taactttttt tttttttttt tttttttttt tttttttttt tacgaatgag gcaatttatt 1954aacccagcat ggtttgttct aatgcttctt gttggcagct gccacctgtc cggcgattct 2014gtccagatct ctttgtccct gaggtgtcag tttgcggccg ctcgagcatg catctaga 2072 2582 PRT Homo sapiens 2 Met His Ala Arg Ala Ala Ala Ala Leu Trp Leu LeuLeu Leu Leu Leu 1 5 10 15 Pro Arg Thr Arg Ala Asp Glu His Glu His ThrTyr Gln Asp Lys Glu 20 25 30 Glu Val Val Leu Trp Met Asn Thr Val Gly ProTyr His Asn Arg Gln 35 40 45 Glu Thr Tyr Lys Tyr Phe Ser Leu Pro Phe CysVal Gly Ser Lys Lys 50 55 60 Ser Ile Ser His Tyr His Glu Thr Leu Gly GluAla Leu Gln Gly Val 65 70 75 80 Glu Leu Glu Phe Ser Gly Leu Asp Ile LysPhe Lys Asp Asp Val Met 85 90 95 Pro Ala Thr Tyr Cys Glu Ile Asp Leu AspLys Glu Lys Arg Asp Ala 100 105 110 Phe Val Tyr Ala Ile Lys Asn His TyrTrp Tyr Gln Met Tyr Ile Asp 115 120 125 Asp Leu Pro Ile Trp Gly Ile ValGly Glu Ala Asp Glu Asn Gly Glu 130 135 140 Asp Tyr Tyr Leu Trp Thr TyrLys Lys Leu Glu Ile Gly Phe Asn Gly 145 150 155 160 Asn Arg Ile Val AspVal Asn Leu Thr Ser Glu Gly Lys Val Lys Leu 165 170 175 Val Pro Asn ThrLys Ile Gln Met Ser Tyr Ser Val Lys Trp Lys Lys 180 185 190 Ser Asp ValLys Phe Glu Asp Arg Phe Asp Lys Tyr Leu Asp Pro Ser 195 200 205 Phe PheGln His Arg Ile His Trp Phe Ser Ile Phe Asn Ser Phe Met 210 215 220 MetVal Ile Phe Leu Val Gly Leu Val Ser Met Ile Leu Met Arg Thr 225 230 235240 Leu Arg Lys Asp Tyr Ala Arg Tyr Ser Lys Glu Glu Glu Met Asp Asp 245250 255 Met Asp Arg Asp Leu Gly Asp Glu Tyr Gly Trp Lys Gln Val His Gly260 265 270 Asp Val Phe Arg Pro Ser Ser His Pro Leu Ile Phe Ser Ser LeuIle 275 280 285 Gly Ser Gly Cys Gln Ile Phe Ala Val Ser Leu Ile Val IleIle Val 290 295 300 Ala Met Ile Glu Asp Leu Tyr Thr Glu Arg Gly Ser MetLeu Ser Thr 305 310 315 320 Ala Ile Phe Val Tyr Ala Ala Thr Ser Pro ValAsn Gly Tyr Phe Gly 325 330 335 Gly Ser Leu Tyr Ala Arg Gln Gly Gly ArgArg Trp Ile Lys Gln Met 340 345 350 Phe Ile Gly Ala Phe Leu Ile Pro AlaMet Val Cys Gly Thr Ala Phe 355 360 365 Phe Ile Asn Phe Ile Ala Ile TyrTyr His Ala Ser Arg Ala Ile Pro 370 375 380 Phe Gly Thr Met Val Ala ValCys Cys Ile Cys Phe Phe Val Ile Leu 385 390 395 400 Pro Leu Asn Leu ValGly Thr Ile Leu Gly Arg Asn Leu Ser Gly Gln 405 410 415 Pro Asn Phe ProCys Arg Val Asn Ala Val Pro Arg Pro Ile Pro Glu 420 425 430 Lys Lys TrpPhe Met Glu Pro Ala Val Ile Val Cys Leu Gly Gly Ile 435 440 445 Leu ProPhe Gly Ser Ile Phe Ile Glu Met Tyr Phe Ile Phe Thr Ser 450 455 460 PheTrp Ala Tyr Lys Ile Tyr Tyr Val Tyr Gly Phe Met Met Leu Val 465 470 475480 Leu Val Ile Leu Cys Ile Val Thr Val Cys Val Thr Ile Val Cys Thr 485490 495 Tyr Phe Leu Leu Asn Ala Glu Asp Tyr Arg Trp Gln Trp Thr Ser Phe500 505 510 Leu Ser Ala Ala Ser Thr Ala Ile Tyr Val Tyr Met Tyr Ser PheTyr 515 520 525 Tyr Tyr Phe Phe Lys Thr Lys Met Tyr Gly Leu Phe Gln ThrSer Phe 530 535 540 Tyr Phe Gly Tyr Met Ala Val Phe Ser Thr Ala Leu GlyIle Met Cys 545 550 555 560 Gly Ala Ile Gly Tyr Met Gly Thr Ser Ala PheVal Arg Lys Ile Tyr 565 570 575 Thr Asn Val Lys Ile Asp 580 3 1827 DNAHomo sapiens CDS (11)..(1747) 3 agcttccacc atg gcg gcc gcc gcg ctg tggctg ctg ctg ctg ctg ctg 49 Met Ala Ala Ala Ala Leu Trp Leu Leu Leu LeuLeu Leu 1 5 10 ccc cgg acc cgg gcg gac gag cac gaa cac acg tat caa gataaa gag 97 Pro Arg Thr Arg Ala Asp Glu His Glu His Thr Tyr Gln Asp LysGlu 15 20 25 gaa gtt gtc tta tgg atg aat act gtt ggg ccc tac cat aat cgtcaa 145 Glu Val Val Leu Trp Met Asn Thr Val Gly Pro Tyr His Asn Arg Gln30 35 40 45 gaa aca tat aag tac ttt tca ctt cca ttc tgt gtg ggg tca aaaaaa 193 Glu Thr Tyr Lys Tyr Phe Ser Leu Pro Phe Cys Val Gly Ser Lys Lys50 55 60 agt atc agt cat tac cat gaa act ctg gga gaa gca ctt caa ggg gtt241 Ser Ile Ser His Tyr His Glu Thr Leu Gly Glu Ala Leu Gln Gly Val 6570 75 gaa ttg gaa ttt agt ggt ctg gat att aaa ttt aaa gat gat gtg atg289 Glu Leu Glu Phe Ser Gly Leu Asp Ile Lys Phe Lys Asp Asp Val Met 8085 90 cca gcc act tac tgt gaa att gat tta gat aaa gaa aag aga gat gca337 Pro Ala Thr Tyr Cys Glu Ile Asp Leu Asp Lys Glu Lys Arg Asp Ala 95100 105 ttt gta tat gcc ata aaa aat cat tac tgg tac cag atg tac ata gat385 Phe Val Tyr Ala Ile Lys Asn His Tyr Trp Tyr Gln Met Tyr Ile Asp 110115 120 125 gat tta cca ata tgg ggt att gtt ggt gag gct gat gaa aat ggagaa 433 Asp Leu Pro Ile Trp Gly Ile Val Gly Glu Ala Asp Glu Asn Gly Glu130 135 140 gat tac tat ctt tgg acc tat aaa aaa ctt gaa ata ggt ttt aatgga 481 Asp Tyr Tyr Leu Trp Thr Tyr Lys Lys Leu Glu Ile Gly Phe Asn Gly145 150 155 aat cga att gtt gat gtt aat cta act agt gaa gga aag gtg aaactg 529 Asn Arg Ile Val Asp Val Asn Leu Thr Ser Glu Gly Lys Val Lys Leu160 165 170 gtt cca aat act aaa atc cag atg tca tat tca gta aaa tgg aaaaag 577 Val Pro Asn Thr Lys Ile Gln Met Ser Tyr Ser Val Lys Trp Lys Lys175 180 185 tca gat gtg aaa ttt gaa gat cga ttt gac aaa tat ctt gat ccgtcc 625 Ser Asp Val Lys Phe Glu Asp Arg Phe Asp Lys Tyr Leu Asp Pro Ser190 195 200 205 ttt ttt caa cat cgg att cat tgg ttt tca att ttc aac tccttc atg 673 Phe Phe Gln His Arg Ile His Trp Phe Ser Ile Phe Asn Ser PheMet 210 215 220 atg gtg atc ttc ttg gtg ggc tta gtt tca atg att tta atgaga aca 721 Met Val Ile Phe Leu Val Gly Leu Val Ser Met Ile Leu Met ArgThr 225 230 235 tta aga aaa gat tat gct cgg tac agt aaa gag gaa gaa atggat gat 769 Leu Arg Lys Asp Tyr Ala Arg Tyr Ser Lys Glu Glu Glu Met AspAsp 240 245 250 atg gat aga gac cta gga gat gaa tat gga tgg aaa cag gtgcat gga 817 Met Asp Arg Asp Leu Gly Asp Glu Tyr Gly Trp Lys Gln Val HisGly 255 260 265 gat gta ttt aga cca tca agt cac cca ctg ata ttt tcc tctctg att 865 Asp Val Phe Arg Pro Ser Ser His Pro Leu Ile Phe Ser Ser LeuIle 270 275 280 285 ggt tct gga tgt cag ata ttt gct gtg tct ctc atc gttatt att gtt 913 Gly Ser Gly Cys Gln Ile Phe Ala Val Ser Leu Ile Val IleIle Val 290 295 300 gca atg ata gaa gat tta tat act gag agg gga tca atgctc agt aca 961 Ala Met Ile Glu Asp Leu Tyr Thr Glu Arg Gly Ser Met LeuSer Thr 305 310 315 gcc ata ttt gtc tat gct gct acg tct cca gtg aat ggttat ttt gga 1009 Ala Ile Phe Val Tyr Ala Ala Thr Ser Pro Val Asn Gly TyrPhe Gly 320 325 330 gga agt ctg tat gct aga caa gga gga agg aga tgg ataaag cag atg 1057 Gly Ser Leu Tyr Ala Arg Gln Gly Gly Arg Arg Trp Ile LysGln Met 335 340 345 ttt att ggg gca ttc ctt atc cca gct atg gtg tgt ggcact gcc ttc 1105 Phe Ile Gly Ala Phe Leu Ile Pro Ala Met Val Cys Gly ThrAla Phe 350 355 360 365 ttc atc aat ttc ata gcc att tat tac cat gct tcaaga gcc att cct 1153 Phe Ile Asn Phe Ile Ala Ile Tyr Tyr His Ala Ser ArgAla Ile Pro 370 375 380 ttt gga aca atg gtg gcc gtt tgt tgc atc tgt tttttt gtt att ctt 1201 Phe Gly Thr Met Val Ala Val Cys Cys Ile Cys Phe PheVal Ile Leu 385 390 395 cct cta aat ctt gtt ggt aca ata ctt ggc cga aatctg tca ggt cag 1249 Pro Leu Asn Leu Val Gly Thr Ile Leu Gly Arg Asn LeuSer Gly Gln 400 405 410 ccc aac ttt cct tgt cgt gtc aat gct gtg cct cgtcct ata ccg gag 1297 Pro Asn Phe Pro Cys Arg Val Asn Ala Val Pro Arg ProIle Pro Glu 415 420 425 aaa aaa tgg ttc atg gag cct gcg gtt att gtt tgcctg ggt gga att 1345 Lys Lys Trp Phe Met Glu Pro Ala Val Ile Val Cys LeuGly Gly Ile 430 435 440 445 tta cct ttt ggt tca atc ttt att gaa atg tatttc atc ttc acg tct 1393 Leu Pro Phe Gly Ser Ile Phe Ile Glu Met Tyr PheIle Phe Thr Ser 450 455 460 ttc tgg gca tat aag atc tat tat gtc tat ggcttc atg atg ctg gtg 1441 Phe Trp Ala Tyr Lys Ile Tyr Tyr Val Tyr Gly PheMet Met Leu Val 465 470 475 ctg gtt atc ctg tgc att gtg act gtc tgt gtgact att gtg tgc aca 1489 Leu Val Ile Leu Cys Ile Val Thr Val Cys Val ThrIle Val Cys Thr 480 485 490 tat ttt cta cta aat gca gaa gat tac cgg tggcaa tgg aca agt ttt 1537 Tyr Phe Leu Leu Asn Ala Glu Asp Tyr Arg Trp GlnTrp Thr Ser Phe 495 500 505 ctc tct gct gca tca act gca atc tat gtt tacatg tat tcc ttt tac 1585 Leu Ser Ala Ala Ser Thr Ala Ile Tyr Val Tyr MetTyr Ser Phe Tyr 510 515 520 525 tac tat ttt ttc aaa aca aag atg tat ggctta ttt caa aca tca ttt 1633 Tyr Tyr Phe Phe Lys Thr Lys Met Tyr Gly LeuPhe Gln Thr Ser Phe 530 535 540 tac ttt gga tat atg gcg gta ttt agc acagcc ttg ggg ata atg tgt 1681 Tyr Phe Gly Tyr Met Ala Val Phe Ser Thr AlaLeu Gly Ile Met Cys 545 550 555 gga gcg att ggt tac atg gga aca agt gccttt gtc cga aaa atc tat 1729 Gly Ala Ile Gly Tyr Met Gly Thr Ser Ala PheVal Arg Lys Ile Tyr 560 565 570 act aat gtg aaa att gac tagagacccaagaaaacctg gaactttgga 1777 Thr Asn Val Lys Ile Asp 575 tcaatttctttttcataggg gtggaacttg cacagcaaaa acaaacaaac 1827 4 579 PRT Homo sapiens4 Met Ala Ala Ala Ala Leu Trp Leu Leu Leu Leu Leu Leu Pro Arg Thr 1 5 1015 Arg Ala Asp Glu His Glu His Thr Tyr Gln Asp Lys Glu Glu Val Val 20 2530 Leu Trp Met Asn Thr Val Gly Pro Tyr His Asn Arg Gln Glu Thr Tyr 35 4045 Lys Tyr Phe Ser Leu Pro Phe Cys Val Gly Ser Lys Lys Ser Ile Ser 50 5560 His Tyr His Glu Thr Leu Gly Glu Ala Leu Gln Gly Val Glu Leu Glu 65 7075 80 Phe Ser Gly Leu Asp Ile Lys Phe Lys Asp Asp Val Met Pro Ala Thr 8590 95 Tyr Cys Glu Ile Asp Leu Asp Lys Glu Lys Arg Asp Ala Phe Val Tyr100 105 110 Ala Ile Lys Asn His Tyr Trp Tyr Gln Met Tyr Ile Asp Asp LeuPro 115 120 125 Ile Trp Gly Ile Val Gly Glu Ala Asp Glu Asn Gly Glu AspTyr Tyr 130 135 140 Leu Trp Thr Tyr Lys Lys Leu Glu Ile Gly Phe Asn GlyAsn Arg Ile 145 150 155 160 Val Asp Val Asn Leu Thr Ser Glu Gly Lys ValLys Leu Val Pro Asn 165 170 175 Thr Lys Ile Gln Met Ser Tyr Ser Val LysTrp Lys Lys Ser Asp Val 180 185 190 Lys Phe Glu Asp Arg Phe Asp Lys TyrLeu Asp Pro Ser Phe Phe Gln 195 200 205 His Arg Ile His Trp Phe Ser IlePhe Asn Ser Phe Met Met Val Ile 210 215 220 Phe Leu Val Gly Leu Val SerMet Ile Leu Met Arg Thr Leu Arg Lys 225 230 235 240 Asp Tyr Ala Arg TyrSer Lys Glu Glu Glu Met Asp Asp Met Asp Arg 245 250 255 Asp Leu Gly AspGlu Tyr Gly Trp Lys Gln Val His Gly Asp Val Phe 260 265 270 Arg Pro SerSer His Pro Leu Ile Phe Ser Ser Leu Ile Gly Ser Gly 275 280 285 Cys GlnIle Phe Ala Val Ser Leu Ile Val Ile Ile Val Ala Met Ile 290 295 300 GluAsp Leu Tyr Thr Glu Arg Gly Ser Met Leu Ser Thr Ala Ile Phe 305 310 315320 Val Tyr Ala Ala Thr Ser Pro Val Asn Gly Tyr Phe Gly Gly Ser Leu 325330 335 Tyr Ala Arg Gln Gly Gly Arg Arg Trp Ile Lys Gln Met Phe Ile Gly340 345 350 Ala Phe Leu Ile Pro Ala Met Val Cys Gly Thr Ala Phe Phe IleAsn 355 360 365 Phe Ile Ala Ile Tyr Tyr His Ala Ser Arg Ala Ile Pro PheGly Thr 370 375 380 Met Val Ala Val Cys Cys Ile Cys Phe Phe Val Ile LeuPro Leu Asn 385 390 395 400 Leu Val Gly Thr Ile Leu Gly Arg Asn Leu SerGly Gln Pro Asn Phe 405 410 415 Pro Cys Arg Val Asn Ala Val Pro Arg ProIle Pro Glu Lys Lys Trp 420 425 430 Phe Met Glu Pro Ala Val Ile Val CysLeu Gly Gly Ile Leu Pro Phe 435 440 445 Gly Ser Ile Phe Ile Glu Met TyrPhe Ile Phe Thr Ser Phe Trp Ala 450 455 460 Tyr Lys Ile Tyr Tyr Val TyrGly Phe Met Met Leu Val Leu Val Ile 465 470 475 480 Leu Cys Ile Val ThrVal Cys Val Thr Ile Val Cys Thr Tyr Phe Leu 485 490 495 Leu Asn Ala GluAsp Tyr Arg Trp Gln Trp Thr Ser Phe Leu Ser Ala 500 505 510 Ala Ser ThrAla Ile Tyr Val Tyr Met Tyr Ser Phe Tyr Tyr Tyr Phe 515 520 525 Phe LysThr Lys Met Tyr Gly Leu Phe Gln Thr Ser Phe Tyr Phe Gly 530 535 540 TyrMet Ala Val Phe Ser Thr Ala Leu Gly Ile Met Cys Gly Ala Ile 545 550 555560 Gly Tyr Met Gly Thr Ser Ala Phe Val Arg Lys Ile Tyr Thr Asn Val 565570 575 Lys Ile Asp 5 7 DNA Artificial Sequence Kozak′s ConsensusSequence 5 accatgg 7 6 15 DNA Artificial Sequence Adapter containing theKozak′s Consensus Sequence 6 agcttccacc atggc 15 7 15 DNA ArtificialSequence Adapter containing the Kozak′s Consensus Sequence (listed as 5′to 3′) 7 ggccgccatg gtgga 15

What is claimed is:
 1. An isolated DNA encoding a human SMBP, whereinsaid DNA comprises a nucleotide sequence of SEQ ID NO:1, with theexception that a sequence comprising the polythymidine sequence frompositions 1899 to 1935 of SEQ ID NO:1 is deleted from a 3′-region fromposition 1875 in the nucleotide sequence of SEQ ID NO: 1; and the SMBPtranslation product of said isolated DNA is expressed at an elevatedlevel compared to the level of expression of a DNA comprising unmodifiedSEQ ID NO:1.
 2. The DNA of claim 1, wherein said DNA comprises anucleotide sequence of SEQ ID NO:1, with the exception that thepolythymidine sequence from positions 1899 to 1935 of SEQ ID NO: 1 isdeleted.
 3. The DNA of claim 1, wherein said DNA comprises a nucleotidesequence of SEQ ID NO:1, with the exception that the nucleotide sequencefrom positions 1875 to 2072 of SEQ ID NO: 1 is deleted.
 4. An isolatedDNA which consists of the nucleotide sequence of SEQ ID NO:
 3. 5. Anexpression vector which carries the DNA of any one of claims 1 to
 4. 6.A transformed cell, which is obtained by culturing cells transformedwith the expression vector of claim 5 under an appropriate condition, ora cellular membrane fraction thereof.
 7. A process for constructing arecombinant human SMBP, which comprises isolating the recombinant humanSMBP from the transformed cells or the cellular membrane fractionsthereof according to claim
 6. 8. A recombinant human SMBP obtained bythe process of claim
 7. 9. The DNA of any one of claims 1, 2, or 3,wherein the surrounding sequence of the start codon is replaced withKozac sequence of SEQ ID NO:5.
 10. An expression vector which carriesthe DNA of claim 9.